Members of the family of BCL-2-related proteins serve as regulators of programmed cell death, or apoptosis (Cory, 1995 Annu. Rev. Immunol. 13, 513-543; Hockenbery, 1995 Nature 348, 334-336; Nunez et al., 1994Nature 348, 334-336; Reed, 1994. J. Cell Biol. 124, 1-6; Akbar et al., 1993. Immunology Today 14, 526-532). Apoptosis has been shown to be involved in several immune processes, including intrathymic deletion of autoreactive cells, elimination of peripheral T cells during the response to viral and bacterial superantigens and lysis of target cells by cytotoxic T lymphocytes. There is increasing evidence that clonal expansion of antigen-specific T-cells is determined by the relative level of cellular proliferation and apoptosis following TCR ligation (Zinkernagel et al. 1993. Immunol. Rev. 131:199). However, the genetic mechanisms responsible for regulating these response phenotypes are not well understood.
The first gene to be identified which encoded a protein in this family, bcl-2, was cloned from the chromosomal breakpoint oft(14;18)-bearing B-cell lymphomas (Tsujimoto et al., 1984. Science 226:1097) and shown to inhibit cellular susceptibility to apoptosis (Cory, supra).
Several genes with homology to bcl-2 have subsequently been characterized, including the following: Al, which encodes an 80-amino acid protein that is rapidly induced in macrophages in response to GM-CSF or LPS (Lin et al., 1993. J. Immunol. 151, 1979-1988); MCL1, an early response gene in myeloid cell lines which undergo macrophage differentiation (Kozopas et al., 1993. Proc. Natl. Acad. Sci. USA 90, 3516-3520); and Bak, a BCL-2 homologue that may enhance apoptosis (Chittenden et al., 1995. Nature 374:733; Kiefer et al., 1995. Nature 374:736).
The bcl-x gene product, closely related to the BCL-2-related protein family, also protects cells from apoptosis. Analysis of mice deficient in BCL-x has suggested that its function is to support the viability of immature cells during development of the nervous and hematopoietic systems (Motoyama et al., 1995. Science 267, 1506-1510; Ma et al., 1995. Proc. Natl. Acad. Sci. USA 92, 4763-4767). Alternative splicing of human bcl-x may result in at least two distinct BCL-x mRNA species. The predominant protein product (233 amino acids) of the larger BCL-x mRNA, BCL-xL, inhibits cell death upon growth factor withdrawal (Boise et al., 1993. Cell 74, 597-608) and its transgenic expression alters thymocyte maturation leading to increased numbers of mature thymocytes (Chao et al., 1995. J. Exp. Med. 182, 821-828; Grillot et al., 1995. J. Exp. Med. 182, 1973-1983). After co-ligation of CD3 and CD28 in murine T-cells, enhanced BCL-xL expression may confer protection from apoptosis (Boise et al., 1995. Immunity 3, 87-98; Radvanyi et al., 1996. J. Immunol. 156, 1788-1798; Mueller et al., 1996. J. Immunol 156, 1764-177 1). The contribution of other isoforms of this gene to activation-induced death in T-cells is less well-defined (Gonzalez-Garcia et al., 1994. Development 120, 3033-3042; Fang et al., 1994 J. Immunol. 153, 4388-4398). A second human BCL-x isoform, BCL-xS, encodes a smaller protein of 170 amino acids which may enhance apoptosis, suggesting that different members of the BCL-x family may have opposing functions. Additional murine BCL-x isoforms, termed BCL-xxcex2 and BCL-xxcex94TM, have been defined. The xcex2 isoform may inhibit apoptosis in neurons (Gonzalez-Garcia et al., 1995. Proc. Natl. Acad. Sci. U.S.A. 92, 4304-4308) and the xcex94TM isoform may inhibit apoptosis in B-cells (Fang et al., supra).
Several proteins which interact with BCL-2 proteins have also been identified including bax, Nip1, Nip2, Nip 3, bad, and bag-1. These various BCL-2 binding proteins have different effects on apoptosis. For example, bak and bax function as inducers of apoptosis, whereas bag increases the resistance of cells to apoptosis (Farrow and Brown. 1996. Curr. Opin. Genetics and Devel. 6:45).
Despite the apparent importance of BCL-x in development and function of T-cells, none of the BCL-x isoforms described so far displays restricted expression with respect to this lineage; all four isoforms of BCL-x are ubiquitously expressed in a wide variety of tissues (Gonzalez-Garcia et al., 1994. Development 120, 3033-3042; Fang et al., supra). This may be because previous studies have isolated most of BCL-x isoforms (BCL-xL, BCL-xS and BCL-xxcex94TM) after screening cDNA libraries from tissues other than T-cells (Gonzalez-Garcia et al., 1994 supra; Fang et al., supra). The physiologic expression of these BCL-x isoforms is not sufficient to confer resistance to apoptosis following TCR ligation, since they are expressed equally well in apoptotic and non-apoptotic T-cell blasts. Moreover, overexpression of Bcl-xL does not affect thymocyte selection (Grillot et al. 1995. J. Exp. Med. 182:1973).
The present invention is based, at least in part, on the discovery of novel molecules, referred to herein as xe2x80x9cBCL-xxcex3xe2x80x9d nucleic acid and protein molecules. The BCL-xxcex3 molecules of the present invention are useful as modulating agents in regulating a variety of cellular processes.
Analysis of the BCL-xxcex3 protein indicates that it contains a BH1 and a BH2 domain which are found in other BCL-x family members. However, the BCL-xxcex3 protein also contains a novel xcex3 domain (shown in amino acids 185-235 of SEQ ID NO:2, which includes an ankyrin domain, e.g., amino acids 185-217 of SEQ ID NO:2). The xcex3 domain (C-terminal amino acids 185-235) of the deduced BCL-xxcex3 protein lacks homology with the C-termini of previously described murine BCL-xxcex3 isoforms, including BCL-xL, BCL-xxcex2 or BCL-xxcex94TM. Since BCL-xxcex3 does not contain an apparent hydrophobic domain flanked by charged residues it is unlikely to be membrane-bound, similar to the murine BCL-xxcex94TM isoform (Gonzalez-Garcia et al., 1994, supra; Fang et al., supra) but in contrast to both, human and murine, BCL-xL and BCL-xS isoforms (Boise et al., 1993) whose C-termini contain sequences that may serve as membrane-anchoring domains (Chen-Levy et al., (1989) Mol. Cell Biol. 9, 701-710; Hochenbery et al., 1990 348, 334-336; Nguyen et al., 1993 J. Biol. Chem. 268, 25265). The BCL-xxcex3 protein has a calculated molecular weight of approximately 26,122 and migrates at approximately 33 kD. The murine amino acid sequence is shown in Seq. ID No. 2.
In contrast to BCL-xL, BCL-xxcex2, and BCL-xxcex94TM, which are expressed in all tissues tested, including brain, eyes, intestine, kidney, liver, lung, lymph nodes, and thymus, the BCL-xxcex3 isoform was detected selectively in thymus, lymph nodes, lung, and eye, but not in heart, intestine, kidney, liver, or brain. BCL-xxcex3 has been found to be expressed only in T-lymphocytes since its message is detected in lymph nodes from BALB/c control but not from BALB/c nu/nu mice or from Rag-2 deficient mice. BCL-xxcex3 is expressed in the less mature, cortisone-sensitive fraction of thymocytes. In addition, BCL-xxcex3 has not been detected in thymuses from class I or class II MHC-deficient B6 mice, implying that expression of this Bcl-x isoform may normally depend on an interaction between the TCR and MHC/peptide complexes. The fact that BCL-xxcex3 has been detected in double positive thymocytes indicates that it plays a role in thymic selection not played by other BCL molecules. Thus, unlike previously described forms of BCL-x molecules, BCL-xxcex3 proteins of the invention are specifically connected to TCR ligation and are essential for resistance to TCR-dependent apoptosis.
In one aspect, the invention features an isolated nucleic acid molecule comprising a nucleotide sequence encoding a naturally occurring BCL-xxcex3. In one embodiment a BCL-xxcex3 nucleic acid molecule encodes mouse BCL-xxcex3. In another embodiment a BCL-xxcex3 nucleic acid molecule encodes human BCL-xxcex3. In a preferred embodiment an isolated BCL-xxcex3 nucleic acid molecule encodes the amino acid sequence of SEQ ID NO:2.
In one embodiment a BCL-xxcex3 nucleic acid molecule comprises a nucleotide sequence encoding a protein having an amino acid sequence at least 60% homologous to the y domain shown in amino acids 185-235 of SEQ ID NO:2 and having anti-apoptotic activity.
In one embodiment a BCL-xxcex3 nucleic acid molecule is at least 92% homologous to the nucleic acid sequence shown in SEQ ID NO:1 or a complement thereof. In a preferred embodiment, a BCL-xxcex3 nucleic acid molecule comprises the sequence shown in SEQ ID NO: 1.
In another embodiment a BCL-xxcex3 nucleic acid molecule encodes an intracellular protein which is anti-apoptotic and has an ankyrin-like domain.
In another embodiment a BCL-xxcex3 nucleic acid molecule comprises a nucleotide sequence at least 80% homologous to the nucleotide sequence shown in nucleotides 930-1082 of SEQ ID NO:1 or a complement thereof.
In another embodiment a BCL-xxcex3 nucleic acid molecule specifically detects a BCL-xxcex3 nucleic acid molecule relative to a nucleic acid molecule encoding another BCL-x molecule. For example, in one embodiment a BCL-xxcex3 nucleic acid molecule hybridizes under stringent conditions to a nucleic acid molecule comprising the nucleotide sequence shown in nucleotides 930-1082 of SEQ ID NO:1.
In a preferred embodiment an isolated BCL-xxcex3 nucleic acid molecule comprises the coding region of SEQ ID NO:1 or a complement thereof. In another embodiment a BCL-xxcex3 nucleic acid molecule further comprises nucleotides 1083-1384 of SEQ ID NO:1. In yet another embodiment a BCL-xxcex3 nucleic acid molecule further comprises nucleotides 1-164 of SEQ ID NO:1. In a further embodiment a BCL-xxcex3 nucleic acid molecule further comprises one or more of: domain B, represented by SEQ ID NO:3; domain C, represented by nucleotides 1085-1193 of SEQ ID NO:1; domain D, represented by SEQ ID NO:4; and domain E, represented by nucleotides 1194-1384 of SEQ ID NO:1 downstream of the BCL-xxcex3 coding sequence. In yet another embodiment a nucleic acid molecule of the present invention has a transcriptional regulatory sequence comprising nucleotides 1-164 of SEQ ID NO:1, which may be operatively linked to the BCL-xxcex3 coding sequence or a heterologous coding sequence.
In yet another embodiment the invention provides an isolated nucleic acid molecule which is antisense to the coding strand of a BCL-xxcex3 nucleic acid molecule
Another aspect of the invention provides a vector comprising a BCL-xxcex3 nucleic acid molecule. In certain embodiments the vector is a recombinant expression vector. In another embodiment the invention provides a host cell containing a vector of the invention. The invention also provides a method for producing BCL-xxcex3 protein by culturing a host cell of the invention in a suitable medium until BCL-xxcex3 protein is produced.
In another aspect the invention provides isolated or recombinant BCL-xxcex3 proteins. In one embodiment a BCL-xxcex3 protein has an ankyrin-like domain, is intracellular, and is anti-apoptotic. In another embodiment an isolated BCL-xxcex3 protein has (i) an amino acid sequence at least 60% homologous to the xcex3 domain amino acid sequence shown in amino acids 185-235 of SEQ ID NO:2 and (ii) having anti-apoptotic activity. In a preferred embodiment a BCL-xxcex3 protein has the amino acid sequence of SEQ ID NO:2. In another embodiment of the invention a BCL-xxcex3 protein is at least about 83.5% homologous to the protein shown in SEQ ID NO:2.
In another embodiment the invention provides a BCL-xxcex3 fusion protein
In another aspect of the invention, antibodies that specifically bind BCL-xxcex3 protein are provided. In one embodiment, the antibodies of the present invention are monoclonal. In another embodiment the subject antibodies are polyclonal.
In another aspect, the invention provides a nonhuman transgenic animal which contains cells carrying a transgene encoding BCL-xxcex3 protein. In one embodiment the transgene alters an endogenous gene encoding endogenous BCL-xxcex3 protein.
In another aspect the present invention provides a method for detecting the presence of BCL-xxcex3 activity in a biological sample by contacting the biological sample with an agent capable of detecting an indicator of BCL-xxcex3 activity such that the presence of BCL-xxcex3 activity is detected in the biological sample. In one embodiment the agent detects BCL-xxcex3 mRNA, e.g., a labeled nucleic acid probe capable of hybridizing to BCL-xxcex3 mRNA. In another embodiment the agent detects BCL-xxcex3 protein, e.g., a labeled antibody that specifically binds to BCL-xxcex3 protein.
In another aspect, the invention provides a method for modulating BCL-xxcex3 activity in a cell comprising contacting the cell with an agent that modulates BCL-xxcex3 activity such that BCL-xxcex3 activity in the cell is modulated. In one embodiment, the agent inhibits BCL-xxcex3 activity. In another embodiment, the agent stimulates BCL-xxcex3 activity. In a preferred embodiment an agent modulates apoptosis in a cell. In one embodiment the agent is an antibody that specifically binds to BCL-xxcex3 protein. In another embodiment the agent modulates transcription of a BCL-xxcex3 gene or translation of a BCL-xxcex3 mRNA. In yet another embodiment, the agent is a nucleic acid molecule having a nucleotide sequence that is antisense to the coding strand of the BCL-xxcex3 mRNA or the BCL-xxcex3 gene.
In one embodiment, the methods of the present invention are used to modulate apoptosis in a T cell. Such methods can be used, e.g., to treat an immune system disorder. In one embodiment BCL-xxcex3 activity is downmodulated to ameliorate an autoimmune disorder. In another embodiment BCL-xxcex3 activity is upmodulated to ameliorate an immunodeficiency.
The present invention also provides a diagnostic assay for identifying a cell or cells at risk for apoptosis in a cell sample, the presence or absence of a genetic lesion characterized by at least one of (i) aberrant modification or mutation of a gene encoding a BCL-xxcex3 protein, and (ii) mis-regulation of said gene; (iii) aberrant post-translational modification of a BCL-xxcex3 protein, wherein a wild-type form of said gene encodes an protein with a BCL-xxcex3 anti-apoptotic activity.
In another aspect the invention provides a method for identifying a compound that modulates the anti-apoptotic activity of a BCL-xxcex3 protein, by providing a indicator composition comprising a BCL-xxcex3 protein having BCL-xxcex3 anti-apoptotic activity, contacting the indicator composition with a test compound, and determining the effect of the test compound on BCL-xxcex3 anti-apoptotic activity in the indicator composition to identify a compound that modulates the anti-apoptotic activity of a BCL-xxcex3 protein.
Other features and advantages of the invention will be apparent from the following detailed description and claims.